This invention relates to a chamber for the preparation of specimens at reduced temperature "cryopreparation," particularly for microscopic or electron microscopic investigation.
In the cryopreparation of specimens, particularly in the cryopreparation of biological specimens for microscopic and electron microscopic studies, preparation chambers are needed to an increasing degree. Such chambers keep both the specimen and the tool at reproducible low temperatures, without icing as a result of the precipitation of atmospheric moisture. Because of the relatively high costs of the liquefied gases traditionally used as coolants ("cryogens") (particularly liquefied nitrogen=N.sub.2 fl or liquefied helium=He.sub.fl), the consumption of cryogen should be kept as small as possible. Finally, the cooled chamber volume should be of such dimensions that both the object in its holder as well as the necessary tools (knives, cutters, saws) and manipulation aids have adequate space in the chamber and can stay constantly in the chamber for synchronous or successive use, without interference with each other. During the preparation of biological specimens using known recrystallization phenomena of aqueous mixed phases and the development of heat associated with machining, chamber temperatures below -150.degree. C. are required. Similar temperatures are needed for machining, particularly for the preparation of sections of viscoelastic plastics with very low glass points (for example, polytetrafluoroethylene). The chamber volume should be at least one liter as a rule, with consideration of the large number of different devices.
According to the state of the art, cold chambers are made of thermally insulating plastics. For example, for taking slices from a specimen, the knife is usually fastened on the slide support of an ultramicrotome (SE Pat. No. 328 426). The knife holder is fastened to the bottom of the chamber for frozen sections. The specimen fastened on the specimen carrier arm of the ultramicrotome is introduced into the cold chamber through an opening in the rear wall, where a thin plastic film seals the opening without hindering the motion of the specimen arm necessary for the cutting. The top opening is covered over entirely or almost entirely by a cover of transparent plastic which permits observation that is absolutely necessary for manipulation. The specimen and the knife are cooled separately by a stream of cold nitrogen gas and electronic feedback control circuits make possible the control of the knife and specimen temperatures by adjusting the nitrogen flow and/or by variation of the power to heaters (cf. "Microscopy," Vol. 25 (1969), pages 17-32).
In another well known arrangement, the specimen is introduced into the cold chamber from above, while the walls consist of insulating plastic and the upper opening is not covered. A continuous adjustable flow of cold nitrogen gas from a Dewar flask regulates the temperature of the chamber atmosphere in this case, according to the signals from a temperature sensor located in the chamber. However, this simple system does not make possible a direct and/or separate preselection of the knife and specimen temperatures, which is required for several technical reasons (cf. "The Journal of Cell Biology," Vol. 51 (1971), pages 772 ff.).
It is common to most known devices that setting the temperature of the specimen and the tool (for example, the knife) is held constant by a feedback control circuit which constantly changes other decisive parameters and introduces disturbances to the system ("control oscillations"). Beyond this, the cooling is generally accomplished by gaseous nitrogen which previously had been liberated by a source of heat in the Dewar flask or recently also by a vaporizer in the cold chamber. The most significant cooling reservoir of liquefied gases, the heat of vaporization, is squandered and the consumption of the cryogen is considerable. Furthermore, the specimen and the knife in many cases are colder than the chamber atmosphere and the chamber wall. For this reason, ice forms preferentially both, since they are the "cooling poles" within the chamber, and on the outer wall of the chamber. If the cold chamber is used in combination with an ultramicrotome to prepare ultrathin slices, both the icing of the chamber and the discharge of cold nitrogen gas from the chamber interfere with the ultramicrotome parts, since changes of length are caused by the resulting temperature changes and these have a substantial effect on the uniformity of a series of slices. In chambers of the construction used for a long time, the very low temperatures (below -150.degree. C.) required for biological cryopreparations cannot be reached or can be reached only for a very short time by the use of a very high consumption of gaseous nitrogen as a coolant.